In cytodiagnostics and in pathology, stained samples are analysed by means of a microscope, generally in transmitted light bright field illumination. The colour of the microscopically analysed sample is an important criterion for diagnosis. In other microscopic analyses, for example by contrast methods such as phase contrast or differential interference contrast (DIC), the colour of the sample is of less significance. In contrast methods of this type, unstained samples are analysed, and appear predominantly transparent in transmitted light bright field microscopy. The contrast methods are subsequently used to make phase properties of the sample visible.
Fluorescence microscopy is a further known analysis method. In this context, the sample which is to be analysed is illuminated by means of an incident light illumination beam path, which passes through what is known as an excitation filter. The excitation light leads to fluorescences in the object which is stained with fluorochromes, the radiated fluorescence light determining the resulting microscope image of the sample. These microscopy methods have been known per se for a relatively long time. For further details, reference is made to the available prior art.
In the last few decades, halogen lamps have been used as the illumination means in the microscope, for example for transmitted light bright field illumination. The light which is emitted by the halogen lamp predominantly corresponds to the continuous spectrum of a black body. Generally, a thermal protection filter is also built into a lighting module comprising a halogen lamp, and greatly attenuates the infra-red range of the emitted radiation. An absorption glass (KG1 having 2 mm thickness) is often used as a thermal protection filter. The continuous spectrum of the resulting illumination makes a reliable colour assessment by the user possible.
In the case of illumination with a particular light source, what is known as the colour rendering index (CRI) is of importance for assessing colours. This is understood to be a photometric value which can be used to describe the quality of the colour rendering of light sources of equal correlated colour temperature. Up to a colour temperature of 5000 K, the light be emitted from a black body of the corresponding colour temperature is used as a reference for assessing the rendering quality. Above a colour temperature of 5000 K, a daylight-like spectral distribution is used as a reference. For example, for calculating the colour rendering of a household filament bulb, which is itself a good approximation to a black body, the spectrum of a black body having a temperature of 2700 K is used. Any light source which perfectly imitates the spectrum of a black body of equal (correlated) colour temperature in the range of the visible wavelengths achieves a colour rendering index of 100. Halogen lamps, similarly to filament bulbs, can achieve colour rendering indices of up to 100.
In microscopy, the halogen lamp is increasingly being replaced with light-emitting diodes (LEDs in the following), which have known advantages. These advantages include greater light radiation at a lower consumption of electrical power and a longer service life. For transmitted light illumination, white LEDs are predominantly used. In a white standard LED, a blue, violet or UV LED is combined with photoluminescent material. Use is generally made of a blue LED, which is combined with a yellow luminescent material. UV LEDs comprising a plurality of different luminescent materials (generally red, green and blue) may also be used. In accordance with the principles of additive colour mixing, white light is produced by LEDs of this type. The components manufactured in this manner have good colour rendering properties, the colour rendering indices being between 70 and 90. However, the white LEDs do not emit a continuous spectrum. White light LEDs which are based on blue LEDs have a strong emission in the blue spectral range (at approximately 450 nm), a minimum in the blue-green (at approximately 500 nm) and a wider emission range up to higher wavelengths, with a maximum at approximately 550 nm, which decreases considerably at approximately 650 nm.
Depending on the type of LED, the ratio of the intensity minimum at 500 nm to the intensity maximum at approximately 450 nm is typically approximately 10-20%. With a discontinuous spectrum of this type as the sample illumination, the colour assessment is more difficult, and differs from the empirical values obtained in the case of microscope illumination by means of a halogen lamp.
DE 10 2007 022 666 A1 addresses this problem. In this document, the illumination for microscopy from a conventional halogen light source, which is combined with a daylight filter, is compared with that of a white light LED. It is found that the colours of the observed object, which is observed either visually or by way of a (CCD) camera, are altered as a result of the different spectral distributions, and this colour alteration can lead to incorrect diagnostic results. In this document, it is therefore attempted to adapt the spectral distribution of a white light LED to the spectral distribution of daylight by means of a so-called “wavelength distribution conversion element”. In this document, several examples of suitable spectral transmission profiles for possible correction filters (“wavelength distribution conversion elements”) are provided, there being the possibility of placing two correction filters in succession. A first example of white light LEDs which are used therein exhibits a first maximum in the blue range (at approximately 450 nm) and a second, in this case higher maximum in the green-yellow range (at approximately 550 nm). By contrast, another example of a white light LED exhibits the spectral profile which was described above in the introduction to the description, in which the first maximum has a higher intensity than the second maximum. In both cases, the respective correction filter provides a spectral profile which still corresponds as a whole to the profile of the original white light LED, but in which the two maxima are adjusted to approximately the same intensity. The resulting spectral profile is thus still a long way away from the desired aim, which is a spectrum of daylight or of a halogen lamp (comprising a daylight filter).
A further problem occurs in fluorescence microscopy, which was addressed in the introduction above. If, in addition to the transmitted light bright field illumination which was discussed above, the microscope additionally has the possibility of incident light fluorescence illumination, the inventors found the following effect. A large proportion of the excitation light which is produced during the incident light fluorescence excitation of the sample passes through the sample and reaches the transmitted light illumination source along the transmitted light illumination axis. If a white light LED comprising a blue LED is arranged at this location, blue excitation light leads to excitation of the yellow-green conversion dye in the white LED, in such a way that, in turn, yellow-green light reaches the sample along the transmitted light illumination axis. This is perceived as a disruptive background in the fluorescence image, and can even overlap considerably with the actual fluorescence from the sample. Analogous effects are found when white light LEDs which are based on violet or UV LEDs are used, if the excitation light of the fluorescence illumination has spectral components in the violet or UV spectral range. In this case, the corresponding conversion dyes in the white light LED are excited. This excitation takes place even when the white light LED per se is switched off.